P1343: TNIK SIGNALLING IMPAIRS SELF-RENEWAL AND LONG-TERM RECONSTITUTION CAPACITY OF HSCS
Authors: Luca Palombella, Michael Amrein, Elias Bührer, Carsten Riether, Adrian Ochsenbein
Journal: HemaSphere (2023)

Topic: 23. Hematopoiesis, stem cells and microenvironment Background: The hematopoietic system is organized hierarchically. Hematopoietic stem cells (HSCs) reside at the top of the hierarchy and have the capacity to self-renew and to differentiate into progenitor cells that give rise to differentiated blood cells. In case of a hematopoietic stress (e.g. infections, inflammation, ionizing radiation etc.) HSCs are able to adapt rapidly to increased demands with increased cell cycling and differentiation. TRAF2 and NCK-interacting kinase (TNIK) belongs to the Ste20 kinase family and was previously shown to activate JNK, NF-κB and Wnt pathways, which all are involved in the regulation of stem cell function. The specific role of TNIK in the hematopoietic system and HSCs has not been studied so far. Aims: To investigate the role of TNIK in the regulation of normal and stress-induced hematopoiesis as well as in HSC function. Methods: A TNIK-deficient mouse model was established in our lab. Flow cytometry was used for immunophenotyping of HSCs, hematopoietic progenitors and differentiated blood cells in TNIK-proficient and -deficient mice in steady-state conditions and after injection of the chemotherapeutic agent 5-fluorouracil (5-FU)). In addition, TNIK mRNA expression was analyzed by qPCR in the different hematopoietic cell subsets. To investigate HSC function, cell cycle analysis by flow cytometry and in vitro colony formation assays were performed. The reconstitution capacity of HSCs was analyzed by competitive bone marrow (BM) transplantations before and after 5-FU injection. Lastly, RNA sequencing analysis was performed to determine the signaling cascades triggered by TNIK in HSCs. Results: TNIK was expressed in HSCs and all progenitor cell subpopulations in the BM of wild-type mice. The hematopoietic stem and progenitor cells composition in the BM of TNIK-/- mice was not altered under steady state condition. Cell cycle analysis showed no difference in HSCs and progenitors in TNIK-/- mice. Similarly, colony forming capacity was not altered in vitro over several replatings. In contrast, competitive transplantation experiments in vivo showed that TNIK-/- HSCs have an increased potential for long-term engraftment. Furthermore, after 5-FU treatment the BM cellularity was higher in TNIK-/- mice compared to wt mice whereas the frequency of HSCs was lower. Competitive transplantation experiments of 5-FU treated BM revealed reduced short-term reconstitution capacity of TNIK-/- but showed no differences in long-term engraftment. Transcriptomic analysis revealed several altered genes involved in the regulation of long-term reconstitution capacity, cell cycling and stress response, such as upregulation of Cyp11a1 and downregulation of Fos and Dusp1 in TNIK-/- HSCs versus TNIKwt HSCs. Summary/Conclusion TNIK deficient HSCs have an improved self-renewal capacity and improved long-term reconstitution capacity. Mechanistically this may be due to the upregulation of Cyp11a1 and down-regulation of Fos. Keywords: Hematopoiesis, Hematopoietic stem cell, Hematopoietic stem and progenitor cells